The most well studied models for gene therapy involve gene transfer using recombinant pathogenic viruses to express new genetic information in order to correct disease phenotypes. Until recently, the most widely researched viral vectors for use in gene therapy were the retroviruses (Miller, A.D., 1990, Human Gene Ther. 1:5-14). A number of difficulties are associated with retroviral use, including the random integration of retroviruses into the host genome which may lead to insertional mutagenesis, or the inadvertent activation of protoncogene expression due to the promoter activity associated with retroviral LTRs (long terminal repeats). Recent evidence using retrovirus vectors in non-human primates has resulted in T cell lymphomas. Efforts in the field of gene therapy have more recently concentrated on the development of viral vectors lacking these particular characteristics.
AAV can assume two pathways upon infection into the host cell. In the presence of helper virus, AAV will enter the lytic cycle whereby the viral genome is transcribed, replicated, and encapsidated into newly formed viral particles. In the absence of helper virus function, the AAV genome will integrate as a provirus into a specific region of the host cell genome through recombination between the AAV termini and host cell sequences (Cheung, A. et al., 1980, J. Virol. 33:739-748; Berns, K. I. et al., 1982, in Virus Persistence, eds. Mahey, B. W. J., et al. (Cambridge Univ. Press, Cambridge), pp. 249-265).
Characterization of the proviral integration site and analysis of flanking cellular sequences indicates specific targeting of AAV viral DNA into the long arm of human chromosome 19 (Kotin, R. M. et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2211-2215; Samulski, R. J. et al., 1991, EMBO J. 10:3941-3950). This particular feature of AAV reduces the likelihood of insertional mutagenesis resulting from random integration of viral vector DNA into the coding region of a host gene. Furthermore, in contrast to the retroviral LTR sequences, the AAV ITR (inverted terminal repeat) sequences appear to be devoid of transcriptional regulatory elements, reducing the risk of insertional activation of protooncogenes.
The AAV genome is composed of a linear single stranded DNA molecule of 4680 nucleotides which contains major open reading frames coding for the Rep (replication) and Cap (capsid) proteins. Flanking the AAV coding regions are two 145 nucleotide inverted terminal (ITR) repeat sequences that contain palindromic sequences that can fold over to form hairpin structures that function as primers during initiation of DNA replication (FIG. 1). Furthermore, experimental observations indicated that the ITR sequences were needed for viral integration, rescue from the host genome and encapsidation of viral nucleic acid into mature virions [Muzyczka, N. 1992, Current Topics in Microbiology & Immunology. 158, 97-29].
Recent work with AAV has been facilitated by the discovery that AAV sequences cloned into prokaryotic vectors are infectious [Samulski, et al. 1982, Proc. Natl. Acad. Sci. U.S.A. 79:2077-2081]. When a plasmid containing intact AAV genome is transfected into cells in the presence of helper virus, AAV can be rescued out from the plasmid vector and enter the lytic pathway leading to production of mature virions. In the absence of helper virus the recombinant AAV vector will integrate into the host cell genome and remain as a provirus until the cell subsequently becomes infected with a helper virus.